Efficacy of optimal nutraceutical combination in treating PCOS characteristics: an in-silico assessment

Background Polycystic ovary syndrome (PCOS) is a serious health condition affecting women of reproductive age. High prevalence of PCOS and associated metabolic complications needs effective treatment and management. This study evaluated the efficacy of optimal nutraceutical combinations in improving PCOS characteristics using system biology-based mathematical modelling and simulation. Methods A shortlisting of eight potent nutraceuticals was carried out with literature search. Menstrual cycle model was used to perform simulations on an in-silico population of 2000 individuals to test individual and combined effects of shortlisted nutraceuticals on five PCOS characteristics [oligomenorrhea, anovulation, hirsutism, infertility, and polycystic ovarian morphology (PCOM)] for a duration of 6 months. Efficacy was tested across lean and obese phenotypes and age groups. Results Individual assessment of nutraceuticals revealed seven most potent compounds. Myo-inositol among them was observed to be the most effective in alleviating the PCOS characteristics. The in-silico population analysis showed that the combination of melatonin and ALA along with myo-inositol was efficacious in restoring the hormonal balance across age-groups and Body Mass Index (BMI) categories. Conclusion Supplementation with the combination of myo-inositol, melatonin, and ALA demonstrated potential in managing PCOS symptoms in our in-silico analysis of a heterogeneous population, including lean and obese phenotypes across various severities and age groups, over a 6-month period. Future clinical studies are recommended to validate these findings. Supplementary Information The online version contains supplementary material available at 10.1186/s12902-024-01571-y.


Background
With a global prevalence of 1.55 million incident cases, polycystic ovary syndrome (PCOS) has become the most common gynaecologic endocrinopathy [1].PCOS is characterized by diverse disorders such as hyperandrogenism, polycystic ovarian morphology (PCOM), oligo-anovulation, insulin resistance-led hyperinsulinemia and dyslipidaemia [2].While the underlying pathological mechanisms are still unclear, it is suggested that insulin resistance (IR) and hyperandrogenaemia (HA) are central to PCOS pathogenesis [3].Insulin resistance-led hyperinsulinemia primarily causes HA and leads to alteration of steroidogenesis in ovary and in adrenal glands [4].Moreover, in PCOS, premature responsiveness to luteinizing hormone (LH) arrests follicular development due to altered steroidogenesis [4].The tendency of granulose cells to secrete anti-Müllerian hormone (AMH), leads to elevated serum concentration of AMH.High circulating AMH desensitizes ovarian follicles to follicle stimulating hormone (FSH), and subsequent premature follicular arrest [5].
PCOS not only lead to impaired fertility but also cause adverse sequelae.Women and adolescent teenagers with PCOS also suffer from metabolic syndrome [6].PCOS is observed to evolve from a reproductive disease to a more metabolic disorder as the age advances [7].Abdominal obesity, lipid disorders, diabetes mellitus, hypertensive and cardiovascular disorders, and endometrial cancer may appear as long-term consequences [7].Therefore, the rising prevalence of PCOS is a concern.Incidence rate of PCOS has increased by 1.45% from 2007 to 2017 globally [1].Various studies from India have estimated prevalence of PCOS between 3.7 and 22.5% [8] with majority of cases being reported from urban India [9].As per an observational study conducted by Bahadur et al. (2020), PCOS usually manifests as ovulatory dysfunction, PCOM and HA in Indian reproductive age women [10].
In most cases, PCOS is diagnosed only when clinical complications appear.These complications consist of menstrual irregularities, infertility, hirsutism, acne, and alopecia [2] which altogether reduces the quality of life in patients [11].Early intervention is hence needed to attenuate adverse outcomes related to PCOS [12].
At present, PCOS management prioritizes on improving clinical symptoms such as ovulation, hirsutism, menstrual irregularity as well as regulating sexual hormones and insulin levels.Currently, metformin and combined oral contraceptives are used as the primary treatment option because of their proven effectiveness in alleviating disease symptoms [13].However, the consumption of metformin is associated with gastrointestinal side effects, vomiting, and nausea [14].Besides, longitudinal followup studies observe high dropout rates during metformin treatment [15].Such shortcomings of conventional treatments have led to an increase in invitro and in vivo research of potential nutraceuticals.Nutraceuticals broadly refer to biologically active components typically found in food, but presented in a non-food format (usually as dietary supplements) with the intention of promoting and improving health [16].They have come across as promising pharmacological agents in improving insulin resistance, disordered lipid profile, inflammation, and related disruptions in molecular pathways involved in PCOS [17].
The present study aims to identify an effective combination of nutraceuticals in PCOS symptom management using system biology-based mathematical modelling and simulation.In-silico quantitative analysis allows a focused approach to identify the potential compounds based on published data.Model-based validation of the effect of these identified nutraceuticals on PCOS severity enable prior impact assessment that can reduce the clinical trial costs.It was hypothesized that the derived optimal combination of nutraceuticals would improve the PCOS symptoms in a model-simulated PCOS population.

Shortlisting of compounds to identify potential nutraceutical combinations
For the purpose of this study, we defined nutraceuticals as vitamins, vitamin-like compounds or polyphenolic compounds having a role in alleviating PCOS symptoms.Eight efficient nutraceutical compounds were identified from the published literature and data analysis (Inositol, vitamin E, coenzyme Q, N-acetyl cysteine, alpha-lipoic acid (ALA), L-carnitine, melatonin and Silybin).These compounds are reported to address most of the known symptoms and physiological perturbations of PCOS.Details of the shortlisting process are mentioned in the supplementary file (Fig. S1).The nutraceuticals were shortlisted by filtering out the compounds which had insufficient data [17][18][19][20][21][22] or compounds associated with potential side-effects [23][24][25][26][27][28].Few compounds were eliminated from investigation due to uncertain efficacy [29][30][31][32][33] and non-reproducible results [23,34].The effect of these eight nutraceuticals is assessed on an in-silico PCOS population for their individual and combined effects.

Computation model description
The model is based on menstrual cycle irregularities in PCOS [35].It represents dynamics of the hypothalamuspituitary-ovarian axis (HPO-axis) and distinct stages of ovarian development.The model simulations could predict mass of pre-antral follicle (PrAP) and serum levels of LH, Estradiol (E2), and Testosterone (T) hormones in PCOS condition.In the present study, simulations were performed to capture the nutraceuticals effect on PCOS characteristics when administered individually and in combination.Based on the mechanism of action of nutraceuticals, the associations were made for each model subsystem parameters.Inositol, L-carnitine, and melatonin are observed to improve ovarian function [36][37][38] and have been associated with 'k2' , 'beta' and 'm1' parameters from ovarian subsystems [35].N-acetyl cysteine and coenzyme Q have a role in glucose metabolism [39,40] and are associated with parameter 'cFSHe' from FSH subsystem [35].Vitamin E lowers testosterone levels [40] and has been associated with parameter 'cLHe' from LH subsystem [35].Silybin is known to improve the PCOS-related non-alcoholic fatty liver condition [36] and has been associated with 'epsilon' parameter from ovarian subsystem [35] and ALA is known to improve lipid profile [41] and associated with rFSH parameter [35].To obtain the optimal combination and effective dosage of nutraceuticals, the pharmacokinetic profile of each nutraceutical was simulated for six months through associated parameters for different doses using the model to study the physiological effect of nutraceuticals on PCOS characteristics.

Scoring system of PCOS
Among the five PCOS characteristics i.e., oligomenorrhea, anovulation, hirsutism, infertility and polycystic ovaries, presence of at least 2 characteristics was considered necessary to establish PCOS condition in the model.This criterion is in line with the published literature for PCOS diagnosis [2].The model considered hormonal peak in ovulatory phase for obtaining LH levels while E2 levels were obtained as mean of ovulatory and luteal phase.The length of the menstrual cycle was obtained from the time period between two consecutive LH surges and was used to score oligomenorrhoea and anovulation [42].E2 is known to support ovulation and pregnancy [42].Hence, its levels were used to score infertility.T levels were used to score hirsutism while the mass of PrAP was used to score polycystic ovaries [43].

In-silico population generation
Heterogeneous in-silico population of 2000 women with PCOS were simulated using delay-differential equation model discussed above.The developed population consists of both lean and obese PCOS phenotype of reproductive age-group (18-40 years).The simulated population was categorized into lean and obese PCOS based on serum triglyceride levels.Populations with triglyceride levels less than 150 mg/dl were considered in lean PCOS (46.06%), whereas those having triglyceride levels greater than or equal to 150 mg/dl were considered in obese (53.94%)PCOS.The thresholds for triglycerides were referred from published studies [44].PCOS individuals varied in terms of cycle length, preantral follicle count, insulin resistance and levels of LH, T, E2, AMH and homeostasis model assessment-estimated insulin resistance (HOMA-IR).

Healthy versus PCOS simulation
PCOS state was simulated by perturbing essential pathophysiological parameters.Frequency of LH peaks and the level of variation in two consecutive LH surges were linked to the number of menstrual cycles in 7 months and the length of cycle, respectively (Fig. 1).The sevenmonth duration shows seven cycles in a healthy state and 6 cycles in PCOS state (Fig. 1 and b).Reduced frequency of cycles indicates an increased cycle length (from 30 days to 35 days) leading to oligomenorrhea in PCOS.LH hormone levels were elevated in case of PCOS state (190 IU/L) in comparison to healthy state (120 IU/L).The mass of the pre-antral follicle increased from healthy to PCOS, resulting in multiple immature follicles (Fig. 1c  and d).Also, the testosterone hormone level was high in PCOS compared to the healthy state (Fig. 1e and f ).

Effect of individual nutraceuticals on PCOS characteristics
The individual nutraceutical simulation results were analysed for their effects on five PCOS characteristics as mentioned in the PCOS scoring system.The effect of each nutraceutical has been measured in terms of fold change.Fold change was obtained by calculating ratio between levels of hormones or metabolites in healthy condition and levels of biochemical parameters in PCOS condition.The healthy condition is hence defined as a fold change of 1.All the eight nutraceuticals were analysed for individual effect on the PCOS condition.Compounds which could show effectiveness in treating at least one PCOS characteristic were deemed suitable to be included in the optimal combination for product formulation.
Figure 3 indicates that melatonin was most effective in treating PCOM amongst eight nutraceuticals by reducing the size of the pre-antral follicle.Inositol, NAC and CoQ10 were found to affect the anovulation by reducing the LH surge.Results show that melatonin, Vit E and CoQ10 showed reduced the cycle length thereby improving oligomenorrhea.ALA, silybin and Vit E were found to be effective in treating infertility by improving the levels of estradiol hormone.Results also suggests that nutraceuticals ALA, silybin and Vit E showed improvement in hirsutism by reducing testosterone hormone levels.Out of the eight shortlisted nutraceuticals, only L-carnitine was not able to show significant change in any PCOS characteristic.Therefore, the effective seven nutraceuticals were shortlisted for optimal nutraceutical combination analysis.

Assessment of optimal nutraceutical combination
The shortlisted seven nutraceuticals were grouped into various combinations based on individual simulation results and the possible synergy between the shortlisted compounds which is known from the literature.These combinations were analysed to compare and finalize the six most potent nutraceutical combinations for treating PCOS.
The population analysis with the above-mentioned optimal nutraceutical combination interventions showed a significant (p < 0.0001) improvement in all PCOS characteristics mentioned in the PCOS scoring system.Figure 4 shows the categorization of the population into different age groups and the effect of interventions with the best combination.Non-significant differences in PCOS scores between ages suggest that the use of the best combination can benefit all age groups.
Population analysis was performed for lean and obese individuals with varying testosterone levels.The results shown in Fig. 5 depict that the intervention with optimal nutraceutical combination shows a significant reduction (p < 0.0001) in testosterone levels for both lean (40.76 ng/ dl ± 1.62) and obese (46.08 ng/dl ± 3.13) PCOS populations.The intervention group also showed a reduction in LH and E2 levels.This implies that the best optimal combination may be effective for infertility treatment as these hormones are required for implantation.Tables 1 and 2 show the descriptive statistics for lean and obese PCOS with the best optimal combination.

Discussion
This study presents an in-silico analysis of effects of various nutraceuticals in treating PCOS characteristics.The eight nutraceuticals shortlisted for simulation analysis were based on their maximum benefits and minimum side effects published in the literature.Model simulation analysis for individual nutraceuticals showed seven nutraceuticals effective in treating various PCOS characteristics.Myo-inositol alone showed improvement in all PCOS characteristics except PCOM.Clinical trials have observed folic acid combination with myo-inositol, but not folic acid alone, have resulted in better outcomes for the PCOM characteristic [46][47][48][49].Also, administering the myo-inositol along with the isoforms of inositol such The combination analysis results suggest inositol, melatonin, and ALA as the best optimal combination effective in maintaining a hormonal balance of LH, AMH, E2, and testosterone.Therefore, a formulation comprising the selected ingredients (myo-inositol (2 g), melatonin (2 mg) and ALA (600 mg)) can be beneficial in alleviating the spectrum of PCOS symptoms across the population.Previous studies have shown the effectiveness and synergy of inositol with melatonin [51] and alpha-lipoic acid [52] upon co-administration.The effects of three compounds revealed from the in-silico combination analysis were in line with their mechanisms of actions known from the literature [52,53].follicles causes polycystic ovaries that in turn contributes to elevated basal serum estrogen level [54].Inositol, ALA, and melatonin are together known to reduce insulin resistance, key mechanism in PCOS pathogenesis [53,55].Melatonin restores the normal pulsation of GnRH, thereby indirectly normalizing LH levels.Myo-inositol and melatonin are known to normalize androgen levels, thereby inhibiting formation of polycystic ovarian morphologies [51].Melatonin also has independently been involved in normalizing estrogen levels [53].

Conclusion
In conclusion, the in-silico analysis of the optimal nutraceutical combination [(myo-inositol (2 g), melatonin (2 mg) and ALA (600 mg)] was observed to be a potential treatment for PCOS when supplemented for a duration of 6 months.The strength of our study is the validation of the nutraceutical effects on a large heterogeneous insilico PCOS population.The heterogenous in-silico population has individuals from all reproductive age groups with lean and obese PCOS.Therefore, the efficacy of the optimal combination of nutraceuticals is predicted with higher confidence.One of the limitations of this study is that we assumed a population devoid of any deficiencies of vitamins and minerals.Low levels of micronutrients such as B-vitamins, vitamin D, chromium, magnesium, and selenium are found to have a crucial role in PCOS condition [56,57].Also, unlike clinical trials, modelling simulation cannot assess the physiological synergy of nutraceutical combinations.Future studies hence should focus on clinical validation of the effect and duration of administration for the obtained nutraceutical combination.

Fig. 2 Fig. 1
Fig. 2 Prevalence of PCOS based on different age groups.Pie chart showing the prevalence from (a) published epidemiological data, (b) population generated in-silico for this study using model simulation.Same age groups are depicted in same colour

Fig. 3
Fig. 3 Heatmap comparing the effect of eight shortlisted nutraceuticals on PCOS characteristics.Expression levels (displayed as fold change from healthy condition) are represented by means of a colour scale in which blue and red represent healthy condition and PCOS respectively.PrAP: Pre-antral follicle mass, LH_P: Time between two LH surge

Figure 6
Figure 6 mentions how the optimal nutraceutical combination has a role in alleviating the disrupted HPOaxis.Insulin resistance causes high pulsations of GnRH leading to increased LH activity [54].High LH increases theca cell stimulation, which results in a hyperandrogenic milieu of the ovary.Hyperandrogenism arrests antral follicle development.Accumulation of pre-antral

Table 1
Statistical analysis showing the effect of optimal nutraceutical combination on PCOS phenotypes in the lean PCOS population *Mean (SD)

Table 2
Statistical analysis showing the effect of optimal nutraceutical combination on PCOS phenotypes in the obese PCOS population *Mean (SD)